An observational study of once weekly intravenous ganciclovir as CMV prophylaxis in heavily pre-treated chronic lymphocytic leukemia patients receiving subcutaneous alemtuzumab.

Fifteen consecutive resistant/relapsed chronic lymphocytic leukemia (CLL) patients (median age: 65 years) received alemtuzumab for 16 consecutive weeks. All patients had negative CMV anti genemia at baseline. Five patients received oral acyclovir 800 mg twice a day for CMV prophylaxis and 10 patients got intravenous (iv) ganciclovir 7.5 mg/kg once a week. A total of five CMV reactivations occurred, four in the acyclovir and one in the ganciclovir prophylaxis group. Alemtuzumab was then discontinued and all patients were treated with iv ganciclovir 7.5 mg/kg per day. All patients achieved negative CMV anti genemia after a median of 15 days of therapy. Weekly iv ganciclovir prophylaxis and alemtuzumab treatment were then restarted without any further CMV reactivations. In conclusion, weekly iv ganciclovir appears feasible and effective in preventing CMV reactivation and disease in this setting of high-risk immunocompromised patients, allowing an easier management of a therapy otherwise difficult to be routinely used.

Keywords: Alemtuzumab; cytomegalovirus; ganciclovir; chronic; lymphocytic; leukemia

There is no proven and effective therapy for patients with chronic lymphocytic leukemia (CLL) who have active disease after prior therapies, including fludarabine. These patients have advanced-stage disease with a poor overall survival outlook [1]. Immunotherapy with the monoclonal antibody alemtuzumab offers a promising alternative to chemotherapy in patients with relapsed or refractory CLL, but its intravenous (i.v.) infusion is almost invariably associated with reactions, sometimes of severe grade [2], [3], [4], [5]. Preliminary studies with subcutaneous (s.c.) alemtuzumab have demonstrated similar efficacy with a better side-effect profile [6], [7], [8], [9]. However, the administration of alemtuzumab is associated with profound panlymphopenia (B-cells and T-cells) and deficiencies in cell-mediated immunity and the major concern regarding its routine use still remains the high incidence of opportunistic infections.

Monitoring for the emergence of opportunistic infections and use of appropriate prophylaxis is mandatory when using alemtuzumab clinically [5],[10]. Cytomegalovirus (CMV) reactivation, studied with periodic analysis of antigenemia and PCR, is frequent in heavily pre-treated CLL patients on alemtuzumab therapy, although only sporadic cases of CMV disease have been described. CMV reactivation has been reported to occur typically after 4 – 8 weeks of therapy, when T-cell depletion is profound. CMV reactivation is estimated to occur in 25 – 50% of CLL patients receiving alemtuzumab [5],[11], but the probability of CMV infection rises to more than 80% in susceptible patients receiving alemtuzumab [12] as part of reduced intensity conditioning (RIC) allografting [13]. Mortality of established CMV disease approaches 100%. Increased incidence of parainfluenza virus, respiratory syncytial virus and adenovirus infections following RIC allografts have also been linked with alemtuzumab treatment, but mortality has generally been low [14].

No prospective reports currently provide results of the use of intravenous ganciclovir as prophylaxis of CMV reactivation in CLL patients receiving subcutaneous alemtuzumab. We, thus, explored the safety and efficacy of anti-viral prophylaxis with iv ganciclovir 7.5 mg/kg once a week in this setting of patients at high risk of CMV reactivation.

Starting from February 2003, 15 heavily pre-treated CLL patients with a median age of 65 years (range 49 – 76) were consecutively enrolled. The characteristics of patients are summarized in Table I. All patients had been previously treated with a median number of three lines of chemotherapy (range 2 – 6), including chlorambucil at standard or high dose, an antracycline-based regimen (CDOP), with or without rituximab immunotherapy. At the time of alemtuzumab therapy the median lymphocyte count in peripheral blood was 27 650 µl (range 4500 – 110 000) and median lymphocytic infiltrate of bone marrow was 95.5% (range 50 – 100). Before treatment with alemtuzumab all patients had normal concentrations of the T-lymphocytes sub-sets and immunoglobulin levels. During iv ganciclovir therapy, all patients were routinely monitored with hematological (hemoglobin, white blood cells and platelets) and biochemical assays (AST, ALT, BUN, uricemia, glucose, creatinine, bilirubin) two or three times a week. All subjects gave written informed consent before entering the study. The protocol was approved by the Institutional Ethics Committee and conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

Table I.  Characteristics of patients at study entry.

Subcutaneous alemtuzumab was administered as previously reported [8]. It was administered to the patients starting from 3 mg up to 30 mg three times a week for up to 12 weeks followed by 30 mg weekly for 4 weeks. Trimethoprim/sulfamethoxazole, twice daily, twice weekly was administered starting on day 1 and continued during therapy up to a minimum of 2 months following the discontinuation of alemtuzumab therapy. Anti-microbial prophylaxis consisted of oral fluconazole and ciprofloxacin. Assessment of response was performed 4 weeks after the completion of therapy and thereafter every third month during follow-up. Response rates were determined according to 1996 National Cancer Institute working Group (NCIWG) criteria [15]. Patients were monitored continuously for alemtuzumab infusion-related toxicity. Side effects were graded according to the WHO toxicity criteria.

To prevent CMV disease, we planned two different, consecutive schedules: oral acyclovir 800 mg twice a day and intravenous ganciclovir 7.5 mg/kg once a week. The patients enrolled between February and December 2003 received oral acyclovir 800 mg twice a day as CMV prophylaxis. On the contrary, intravenous ganciclovir 7.5 mg/kg once a week was administered to those patients enrolled after January 2004. The two groups of patients were comparable for age, disease status at the enrollment, prior lines of therapy and performance status. Anti-viral prophylaxis was started on day 1 and was given during therapy and for 1 month after alemtuzumab discontinuation.

Treatment for CMV infection as manifested by positive blood CMV antigenemia was ganciclovir 7.5 mg/kg per day for 2 weeks or until negative blood CMV antigenemia.

CMV antigenemia and serology were assessed in all patients before entering the study. CMV anti genemia was performed twice weekly up to 2 months after alemtuzumab discontinuation. CMV reactivation was monitored by the CMV pp65 anti genemia assay (Light diagnostics CMV pp65 Antigenemia IFA, KIT Chemicon International Inc., Temecula, USA), according to the manufacturer's instructions. Positive results were considered with one or more CMV antigen positive cells per set of triplicate wells (expressed as number of positive cells per 1×106 leukocytes). Samples were referred as not evaluable when there was insufficient leukocyte number to perform the test.

According to Leruez-Ville et al. [16], CMV reactivations were defined as the presence of at least one pp65 antigen-positive cell per 1×106 leukocytes. CMV disease was diagnosed from the association of clinical symptoms with virologic confirmation of CMV infection of an organ through histological samples obtained with the biopsy of the stomach, duodenum, brain, liver, heart or kidney or from bronchoalveolar lavage fluid or cerebrospinal fluid.

'First dose' flu-like symptoms related to alemtuzumab infusion, which are frequently seen after iv administration of alemtuzumab, were observed only in four patients. A local grade I injection side reaction was observed in 70% of cases, during the first week of therapy. No treatment discontinuation was required for any patient. Eleven cases developed grade 3 neutropenia, requiring G-CSF support. During neutropenia, transient fever of unknown origin occurred in six cases. All patients received iv or oral antibiotic therapy and promptly responded. Hospitalization was not required in any patient. No late-occurring infections or other side effects were observed in any patient during the follow-up period.

Complete remissions were achieved in four patients and partial remissions in eight, producing an overall response rate of 80%. Three patients had stable disease. CLL cells disappeared from blood after a median time of 17 days (range 7 – 60). Median time to response was 3.9 months (range 1.6 – 5.3 months). The four patients achieving CR successfully mobilized PBSC with G-CSF following the last dose of alemtuzumab. Three of them were then transplanted with rapid and sustained hematopoietic recovery and are still in CR after 21, 18 and 2 months, respectively. Two patients progressed after an initial PR and were retreated with the same protocol. One achieved a PR whereas another one developed Hodgkin's disease and died 6 months later. With a median follow-up of 31 months, four patients are in CR, seven in PR, two died and two are alive with progressive disease. No late infections or other side effects were observed in any of the patients during the long-term follow-up period.

Five patients received oral acyclovir 800 mg twice a day for CMV prophylaxis and 10 patients got intravenous (iv) ganciclovir 7.5 mg/kg once a week. At baseline, all patients had negative CMV antigenemia. CMV reactivation occurred in five patients: four in the acyclovir and one in the ganciclovir group. CMV reactivation appeared after a median of 4 weeks (range 3 – 6) of alemtuzumab treatment with a median of 3.5 (range 2 – 10) pp65 antigen positive cells per 100 000. The median PB lymphocyte count was 570 µl (range 300 – 850) at the time of CMV reactivation. Alemtuzumab was discontinued in the presence of a documented CMV reactivation and patients were treated immediately with iv ganciclovir 7.5 mg/kg per day. One patient, treated with oral acyclovir for CMV prophylaxis, showed clinical evidence of CMV pneumonia requiring intensive care hospitalization coupled with anti-CMV treatment with iv ganciclovir 7.5 mg/kg per day. At present, he is alive and well, 20 months after CMV pneumonia. After a median of 15 days of daily iv ganciclovir therapy all patients achieved negative CMV anti genemia. Weekly iv 7.5 mg/kg ganciclovir prophylaxis and alemtuzumab treatment were restarted and no further CMV reactivations were observed. Weekly ganciclovir treatment was well tolerated and renal dysfunction, myelotoxicity and other side effects were not observed during this study.

Alemtuzumab is an effective therapy for patients with relapsed or refractory CLL, but it is associated with profound panlymphocytopenia (B-cells and T-cells) and deficiencies in cell-mediated immunity. CMV reactivation is estimated to occur worldwide in 10 – 25% of CLL patients receiving alemtuzumab treatment [5],[17],[18], requiring routine weekly monitoring by CMV antigenemia or qualitative PCR [12]. However, it has been demonstrated that this percentage is quite different in Southern Europe, where CMV reactivation occurs in more than 50% of CLL patients receiving alemtuzumab [11],[18]. The high incidence of CMV reactivation in CLL patients treated with subcutaneous alemtuzumab in Southern Europe was recently stressed in a paper by Montillo et al. [11], who reported a CMV reactivation rate of 53%. As a matter of fact, whether patients showing only CMV antigenemia and/or PCR positivity require specific anti-viral therapy still remains debated. In fact, only sporadic cases of CMV disease occurring after alemtuzumab were reported. Nevertheless, mortality of established CMV disease is very high.

No data are currently available to recommend routine prophylaxis for CMV reactivation and disease [5]. Concerns over mandatory prophylaxis include additional myelosuppression and potential generation of CMV resistance. Conversely, some authors suggest the use of famciclovir, acyclovir or valaciclovir during alemtuzumab therapy and for at least 2 months after discontinuation [10],[19]. Oral valganciclovir seems to be an attractive and safe alternative to intravenous ganciclovir for pre-emptive CMV treatment in T-cell-depleted allo-SCT recipients [20],[21]. However, no data are up to now available about the valganciclovir use for CMV prophylaxis in CLL patients receiving alemtuzumab. Furthermore, it has recently been demonstrated that patients with B-CLL have an increased relative and absolute number of CD8+ cells, which exhibit a CD45RA+CD27− cytotoxic phenotype [22]. The analysis of specific immune responses with tetrameric CMV-peptides complexes showed that patients with B-CLL had significantly increased numbers of tetrameric-binding CMV-specific CD8+ T-cells. Thus, the authors suggested that in patients with B-CLL the T-cell compartment is shifted toward a CD8+ cytotoxic cell type in an effort to control infections with persistent virus such as CMV [22]. In addition, they speculated that this finding may offer an explanation for the high incidence of CMV reactivation in CLL patients treated with T-cell-depleting agents, such as alemtuzumab [22].

We designed an observational study to evaluate the safety and the efficacy of iv ganciclovir 7.5 mg/kg once a week as a prophylaxis of CMV infection in comparison to oral acyclovir administered daily, administered to 10 heavily pre-treated CLL patients receiving subcutaneous alemtuzumab therapy. Weekly iv ganciclovir prophylaxis was well tolerated and renal dysfunction, myelotoxicity and other side effects were not observed. Nausea and vomiting were not different between the two groups, occurring in a low percentage of patients.

CMV reactivations were monitored only by the CMV pp65 antigenemia assay, performed twice weekly up to 2 months after alemtuzumab discontinuation. Five CMV reactivations were observed between the two groups, four (80%) in the acyclovir group (one of which evolving in a CMV pneumonia) and one (10%) in the ganciclovir group. CMV reactivations developed from 3 – 6 weeks of alemtuzumab treatment, during the nadir of CD4 and CD8 cells, independently from the type of anti-viral therapy. Focusing on patients presenting CMV reactivation in the acyclovir group, they were first treated with daily iv ganciclovir 7.5 mg/kg and then with weekly iv ganciclovir when restarting alemtuzumab therapy. None of them developed a new CMV reactivation.

The introduction of iv ganciclovir prophylaxis minimized the incidence of CMV reactivation without any relevant clinical toxicity. In addition, no late infections or other side effects were observed in any of the patients during the long-term follow-up period. This suggests that iv ganciclovir might reduce the risk of CMV reactivation, allowing an easier management of a therapy otherwise difficult to be routinely used. To the best of our knowledge, this is the first observation regarding the possible effectiveness of weekly iv ganciclovir to prevent CMV reactivation in CLL patients during alemtuzumab treatment. Further randomized studies examining routine CMV monitoring vs anti-viral prophylaxis in a larger number of patients are warranted to adequately direct future care of CLL patients submitted to alemtuzumab therapy.

Supported in part by AIL Pesaro Onlus.